Genetic and biochemical studies lend strong support to the idea that the CTR-family of membrane proteins mediate cellular copper uptake. Similarly, the roles of copper transporting ATPases and intracellular copper chaperones in copper secretion and transfer of copper ions to the active sites of cuproenzymes have been firmly established. What remains elusive, is how these different components are integrated at the molecular level, largely because the structures of the membrane proteins involved in these processes are unknown. The longterm goal of this project is to understand at a structural level the mechanism by which copper transporting membrane proteins and cellular copper chaperones interact with each other to accomplish directed flow of copper within cells. Work towards Aim 1 capitalizes on our progress in the structure determination of the human high affinity copper transporter hCTR1. The structure suggests that clustering of the C-termini in the hCTR1 trimer creates a binding site for both copper ions and copper chaperones, which would allow for a direct transfer of copper from hCTR1 to the chaperones. Notably, the amino acid residues forming this site are absent in mammalian CTR2, raising the question whether all CTR-proteins use the same mechanism to distribute copper to downstream acceptors. We propose to solve the structure of hCTR2 by electron crystallography to investigate the hypothesis that in hCTR2 an alternate set of metal binding residues can functionally replace the C-terminal chaperone docking site observed in hCTRl Further pursuing the idea of direct CTR:chaperone interactions, the goal of Aim 2 is to trap CTR:chaperone complexes, and to visualize their structure by electron microscopic single particle methods. Finally, the longterm goal for Aim 3 is to solve the structure of the copper transporting ATPase 7B, which is involved in copper secretion. The key aspect of these studies will be to delineate the spatial relationships between the membrane embedded and cytosolic domains that are critical for function and the regulation of the ATPase's complex trafficking behavior.

Public Health Relevance

Copper biology rapidly emerges as a critical area in the understanding of normal cellular function, metallinked disorders known as Wilson and Menkes Disease, and neuropathologies like Parkinsons, Alzheimer or Creutzfeldt Jacob disease. The proposed studies will yield mechanistic insights into copper transport and will provide clues what enables cellular copper transporters to also shuttle cancer drugs like cisplatin.